WO2009029681A2 - Micro-arn utilisés pour inhiber la réplication virale - Google Patents

Micro-arn utilisés pour inhiber la réplication virale Download PDF

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WO2009029681A2
WO2009029681A2 PCT/US2008/074513 US2008074513W WO2009029681A2 WO 2009029681 A2 WO2009029681 A2 WO 2009029681A2 US 2008074513 W US2008074513 W US 2008074513W WO 2009029681 A2 WO2009029681 A2 WO 2009029681A2
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mir
nucleic acid
hcv
acid molecule
seq
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WO2009029681A3 (fr
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Michael David
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The Regents Of The University Of California
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/10—Production naturally occurring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus

Definitions

  • the present invention relates to reducing accumulation of viral genomes in a target cell.
  • the present invention provides compositions and methods for combating viral infection through RNA interference.
  • the present invention provides cellular microRNA mimics for treating virus-infected cells.
  • HCV Hepatitis C virus
  • HCV hepatitis C virus
  • the virus has the unique ability to cause persistent infection in susceptible hosts after parenteral or percutaneous transmission.
  • the immunologic correlates of protection and viral clearance and the pathogenesis of liver injury are yet to be defined.
  • Nearly 70% to 80% of infected persons become chronic carriers, and chronic and progressive HCV infection carries significant morbidity and mortality (e.g., major cause of cirrhosis, end-stage liver disease, and liver cancer).
  • the present invention relates to reducing accumulation of viral genomes in a target cell,
  • the present invention provides compositions and methods for combating viral infection through RNA interference.
  • the present invention provides cellular microRNA mimics for treating virus-infected cells.
  • the present invention provides methods for reducing accumulation of Hepatitis C virus (HCV) RNA in a target cell, the method comprising: introducing a first isolated nucleic acid molecule into a Hepatitis C virus (HCV)-infected target cell under conditions suitable (e.g., in an amount effective) for reducing accumulation of HCV RNA in the target cell, wherein the first isolated nucleic acid molecule comprises the nucleotide sequence of a cellular microRNA selected from the group consisting of miR-196, miR-296, miR-351, miR-431 and miR-448.
  • the cellular microRNA is a murine microRNA selected from the group consisting of miR-196, miR-296, miR-351, miR-431, and miR-448.while in other embodiments, the cellular microRNA is a human homolog of the murine microRNA. In some embodiments, the cellular microRNA comprises one of the group consisting of a mature microRNA, a pre-microRNA, and a seed sequence of the mature microRNA (e.g., 5' residues 2 to 7 or 2 to 8 of the mature microRNA).
  • the cellular microRNA is the miR-196 consisting of the nucleotide sequence set forth as SEQ ID NO:7, the miR-296 consisting of the nucleotide sequence set forth as SEQ ID NO:8, the cellular microRNA is the miR-351 consisting of the nucleotide sequence set forth as SEQ ID NO:9 ; the miR-431 consisting of the nucleotide sequence set forth as SEQ ID NO: 10 or the miR-448 consisting of the nucleotide sequence set forth as SEQ ID NO: 11.
  • the methods further comprise introducing a second isolated nucleic acid molecule into the HCV-infected target cell, wherein the second isolated nucleic acid molecule comprises the nucleotide sequence complementary to miR-122.
  • the miR-122 consists of the nucleotide sequence set forth as SEQ ID NO:3.
  • the cellular microRNA comprises the miR-196, the miR-296, the miR-351, the miR-431 and the miR-448.
  • one or both of the first and second nucleic acids are part of a composition further comprising one of the group consisting of a cationic lipid, a neutral lipid, and polyethylene glycol (PEG).
  • the methods of the present invention reduce HCV replication in an infected target cell.
  • the target cell is a hepatocyte.
  • the target cell is in vitro, while in other embodiments the target cell is in vivo.
  • the methods comprise administering one or both of the first and second isolated nucleic acid molecules to a subject comprising the HCV-infected target cell. Some methods further comprise treating the subject for an HCV mediated disease condition. In some embodiments, the methods further comprise administering one or both of a type I interferon (e.g., IFN-alpha or IFN-beta) and ribavarin.
  • a type I interferon e.g., IFN-alpha or IFN-beta
  • compositions comprising: an isolated nucleic acid molecule consisting of the nucleotide sequence of one of the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO: 11 ; and a pharmaceutically acceptable delivery vehicle.
  • the present invention also provides compositions comprising: an isolated nucleic acid molecule consisting of 18 to 25 nucleotides having a nucleotide sequence identity of at least 90% to one of the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10 and SEQ ID NO:11; and a pharmaceutically acceptable delivery vehicle.
  • the nucleic acid molecule is a miRNA. In some embodiments, the nucleic acid molecule is single-stranded, while in others the nucleic acid molecule is at least partially double-stranded. In some embodiments, the nucleic acid molecule is selected from the group consisting of RNA, DNA and modified nucleotide molecules. In some preferred embodiments, the nucleic acid molecule is comprised within an expression vector.
  • kits comprising: an isolated nucleic acid molecule comprising the nucleotide sequence of a cellular microRNA selected from the group consisting of miR-196, miR-296, miR-351, miR-431 and miR-448; and instructions for use in practicing methods comprising: introducing said isolated nucleic acid molecule into a Hepatitis C virus (HCV)-infected target cell under conditions suitable (e.g., nucleic acid in an amount effective) for reducing accumulation of HCV RNA in the target cell.
  • the kit further comprises a pharmaceutically acceptable delivery vehicle for the isolated nucleic acid molecule.
  • the interferon up-regulated cellular microRNA is selected from the group consisting of the nucleotide sequences of Figure 5.
  • the virus is an RNA virus
  • the RNA virus is selected from the group consisting of Hepatitis C virus (HCV) 3 dengue virus, human immunodeficiency virus (HIV), and influenza virus.
  • HCV Hepatitis C virus
  • HAV human immunodeficiency virus
  • influenza virus Compositions and kits for practicing the methods of the present invention are provided as well.
  • the present invention provides method comprising: introducing an isolated nucleic acid molecule into a virus-infected target cell under conditions suitable (e.g., nucleic acid in an amount effective) for reducing accumulation of viral nucleic acid in the target cell, wherein the isolated nucleic acid molecule comprises the reverse complement nucleotide sequence of an interferon down-regulated cellular microRNA.
  • the interferon down- regulated cellular microRNA is selected from the group consisting of the nucleotide sequences of Figure 6.
  • the RNA virus is selected from the group consisting of Hepatitis C virus (HCV), dengue virus, human immunodeficiency virus (HIV), and influenza virus.
  • HCV Hepatitis C virus
  • HAV Hepatitis C virus
  • HAV dengue virus
  • HAV human immunodeficiency virus
  • influenza virus Compositions and kits for practicing the methods of the present invention are provided as well.
  • the present invention provides methods comprising: introducing an isolated nucleic acid molecule into a target cell, wherein the isolated nucleic acid molecule comprises the nucleotide sequence of an interferon-modulated cellular microRNA.
  • the interferon modulated cellular microRNA is an interferon up-regulated cellular microRNA selected from the group consisting of the nucleotide sequences of Figure 5.
  • the interferon modulated cellular microRNA is an interferon down-regulated cellular microRNA selected from the group consisting of the nucleotide sequences of Figure 6.
  • the methods of the present invention establish a type I interferon-treated state in the target cell (e.g., state in which at least one cellular microRNA is modulated up or down regulated modulated in a manner resembling treatment of the target cell with a type I interferon (e.g., up or down regulated in direction if not magnitude as shown in Figure 5 or Figure 6).
  • a type I interferon e.g., up or down regulated in direction if not magnitude as shown in Figure 5 or Figure 6
  • the cellular microRNA is a murine microRNA, while in other embodiments the cellular microRNA is a human homolog of the murine microRNA.
  • the cellular microRNA comprises one of the group consisting of a mature microRNA, a pre-microRNA, and a seed sequence of the mature microRNA (e.g., 5' residues 2 to 7 or 2 to 8 of the mature microRNA).
  • the cellular microRNA comprises a plurality of microRNAs (e.g., 2, 3, 4, 5 or more).
  • the target cell is a hepatocyte. In some embodiments, the target cell is in vitro, while in other embodiments the target cell is in vivo.
  • FIGS IA-E show regulation of miR expression by IFN ⁇ in Huh7 cells and primary hepatocytes.
  • Huh7 cells a, or primary hepatocytes b were stimulated with 100 U/ml IFN ⁇ for 2hrs, and levels of the indicated miRs were quantitated by realtime PCR.
  • ISG54 induction is shown for comparison,
  • c Time course of miR induction by IFN ⁇ : Huh7 cells were stimulated with 100 U/ml IFN ⁇ for the indicated times, and miR-1, miR- 196 or ISG54 expression levels were quantitated by real-time PCR.
  • a, JFHl-replicon containing Huh7 cells were transfected with either a single non-specific miR-mimics (miR-Ctl), a pool of control miRs (miR-Ctl(mix5)) or anti- miRs ( ⁇ miR-Ctl), or specific miR-mimics corresponding to the eight IFN ⁇ -induced miRs or specific ⁇ miRs as indicated.
  • miR-Ctl a pool of control miRs
  • ⁇ miR-Ctl(mix5) or anti- miRs
  • specific miR-mimics corresponding to the eight IFN ⁇ -induced miRs or specific ⁇ miRs as indicated.
  • a combination of the five miRs that displayed anti- viral activity individually was used (miR-mix5) with or without anti- miR-122 as indicated, and HCV genomic RNA was quantitated by real-time PCR after 48 hrs.
  • Huh7 cells were infected with live JFH-I virus for 48 hrs (bars represent means+/-std of at least four independent experiments; p-values are derived from paired Student's t-tests).
  • FIG. 3A-3B show IFN ⁇ -induced miRs directly target viral genomic RNA.
  • a and b An infectious chimeric virus was constructed from JFHl and J6CF as shown in (a), and described in the supplemental M&M.
  • the Huh7 cell subclone Huh7.5.1c2 were transfected with either miR-196 or ⁇ u ' R-448, or with the mutant miR-196* and miR-448* harboring a compensatory mutation in the seed sequence as outlined in (a).
  • Transfected Huh7.5.1c2 cells were infected with JFHl D i 8 3 23 or chimeric J6/JFH, and HCV genomic RNA was quantitated by real-time PCR after 24 or 60 hrs post infection, respectively, during the phase of exponential viral RNA amplification, to accommodate the difference in replication kinetics between the two viruses (bars represent means+/-sem of at eight independent experiments; p-values are derived from paired Student's t-tests).
  • FIG. 4 shows IFN ⁇ -induced miRs mediate anti-viral IFN ⁇ responses of against HCV.
  • JFHl-replicon containing Huh7 were transfected with either nonspecific miR-mimics (miR-Ctl), or anti-miRs ( ⁇ miR-Ctl), or a pool of anti-miRs ( ⁇ miR-Ctl(5)), or a combination of ⁇ miR complementary to the five IFN ⁇ -induced miRs with potent anti-viral effect ( ⁇ miR-mix5), and/or with specific miRs and anti- miRs as indicated prior to stimulation with IFN ⁇ for 48 hrs.
  • miR-Ctl nonspecific miR-mimics
  • ⁇ miR-Ctl anti-miRs
  • ⁇ miR-Ctl pool of anti-miRs
  • ⁇ miR-Ctl(5) a combination of ⁇ miR complementary to the five IFN ⁇
  • Figure 5 provides a listing of the interferon up-regulated cellular miRs identified during development of the present invention.
  • Figure 6 provides a listing of the interferon down-regulated cellular miRs identified during development of the present invention. GENERAL DESCRIPTION OF THE INVENTION
  • RNA interference through non-coding microRNAs represents a vital component of the innate antiviral immune response in plants and invertebrate animals, however, a role for cellular miRs in the defense against viral infection in mammalian organisms has thus far remained elusive '.
  • interferon beta IFN ⁇ rapidly modulates the expression of numerous cellular miRs, with eight of these IFN ⁇ -induced miRs having sequence- predicted targets within the hepatitis C virus (HCV) genomic RNA.
  • the present invention provides compositions and methods comprising cellular miR mimics for inhibiting viral replication.
  • the present invention relates to reducing accumulation of viral genomes in a target cell.
  • the present invention provides compositions and methods for combating viral infection through RNA interference.
  • the present invention provides cellular microRNA mimics for treating virus-infected cells.
  • the present invention provides methods of reducing the amount of a viral genome in a target cell, where the target cell may be present in vitro or in vivo.
  • the term "reducing the amount of indicates that the level or quantity of the viral genome in the target cell is reduced by at least about 2-fold, usually by at least about 5-fold (e.g., 10-fold, 15-fold, 20-fold, 50-fold, or 100-fold or more) as compared to a control (e.g., an untreated target cell).
  • a miRNA (microRNA) mimic is introduced into the target cell, where any convenient protocol for introducing the agent into the target cell may be employed.
  • the miRNA mimic is an agent that reproduces the antiviral effects of IFN ⁇ -induced cellular miRNAs in the target cell.
  • miRNAs are single stranded RNA molecules that range in length from about 20 to about 25 nt (e.g., 20, 21, 22, 23, 24 or 25 nt).
  • the miRNA mimics may or may not be completely complementary to a region of the same length as in the target viral genome. If not completely complementary, the miRNA and its corresponding target viral genome are at least substantially complementary, such that at least six, seven or eight matches are present in the "seed region" near the 5' end of the miR-RNA heteroduplex.
  • RNAi mimics include, but are not limited to: antisense oligonucleotides, such as the specific antisense oligonucleotides reported in the experimental section below, and the like. Also of interest in certain embodiments are RNAi agents. In representative embodiments, the RNAi agent targets the viral RNA genome.
  • RNAi agent refers to an agent that modulates expression of viral RNA by a RNA interference mechanism.
  • RNAi agents employed in one embodiment of the subject invention are small at least partially double-stranded ribonucleic acid molecules, also referred to herein as interfering ribonucleic acids, (e.g., oligoribonucleotides present in duplex structures such as two distinct oligoribonucleotides hybridized to each other or a single ribooligonucleotide that assumes a hairpin formation).
  • interfering ribonucleic acids e.g., oligoribonucleotides present in duplex structures such as two distinct oligoribonucleotides hybridized to each other or a single ribooligonucleotide that assumes a hairpin formation.
  • oligoribonucleotide is meant a ribonucleic acid that does not exceed about 100 nt in length, and typically does not exceed about 75 nt length (e.g., no longer than 70 nt, 65 nt, 60 nt, 55 nt, 50 nt, 45 nt, 40 nt, 35 nt, 30 nt, 25 nt, 20 nt or 15 nt).
  • the length of the duplex structure typically ranges from about 15 to 30 bp, where lengths between about 20 and 25 bps (e.g., 20 bp, 21 bp, 22 bp, 23 bp, 24 bp or 25 bp), are of particular interest in certain embodiments.
  • the RNA agent is a duplex structure of a single ribonucleic acid that is present in a hairpin formation, a shRNA
  • the length of the hybridized portion of the hairpin is typically the same as that provided above for the siRNA type of agent or longer by 3 to 9 nucleotides,
  • miRNA mimics useful for modulating HCV gene expression by RNA interference include short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules, including cocktails of such small nucleic acid molecules and lipid nanoparticle formulations.
  • the miRNA mimic is a locked-nucleic-acid-modified oligonucleotide (e.g., LNA-miR and/or LNA-antimiR), which can be formulated in a physiologically acceptable buffer for intravenous injection (0.01 to 10 mg/kg).
  • the RNAi agent may encode an interfering ribonucleic acid.
  • the RNAi agent may be a transcriptional template of the interfering ribonucleic acid.
  • the transcriptional template is typically a DNA that encodes the interfering ribonucleic acid.
  • the DNA may be present in a vector, where a variety of different vectors are known in the art (e.g., a plasmid vector, a viral vector, etc.).
  • the miRNA mimic can be introduced into the target cell(s) using any convenient protocol, where the protocol will vary depending on whether the target cells are in vitro or in vivo. Where the target cells are in vivo, the miRNA agent can be administered to the host using any convenient protocol.
  • the protocol employed is typically a nucleic acid administration protocol. The following discussion provides a review of representative nucleic acid administration protocols that may be employed.
  • the nucleic acids may be introduced into tissues or host cells by any number of routes, including viral infection, microinjection, or fusion of vesicles.
  • Jet injection may also be used for intramuscular administration (Furth et al., Anal Biochem 205:365-368, 1992).
  • the nucleic acids may be coated onto gold microparticles, and delivered intradermally by a particle bombardment device, or "gene gun” as described in the literature (Tang et al., Nature 356:152-154, 1992), where gold microprojectiles are coated with the DNA, then bombarded into skin cells.
  • Expression vectors may be used to introduce the nucleic acids into a cell. Such vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences.
  • Transcription cassettes may be prepared comprising a transcription initiation region, the target gene or fragment thereof, and a transcriptional termination region.
  • the transcription cassettes may be introduced into a variety of vectors such as plasmids or viruses (e.g., retrovirus, lentivirus, adenovirus, and the like), where the vectors are transiently or stably maintained in the transfected cells, usually for a period of at least about one day, more usually for a period of several days to several weeks.
  • vectors such as plasmids or viruses (e.g., retrovirus, lentivirus, adenovirus, and the like), where the vectors are transiently or stably maintained in the transfected cells, usually for a period of at least about one day, more usually for a period of several days to several weeks.
  • the miRNA mimic can be fed directly to or injected into the host organism containing a target gene (e.g., a target cell infected by a virus).
  • the miRNA may be directly introduced into the cell (e.g., intracellularly), or introduced extracellularly into a cavity, interstitial space, or into the circulation of an organism.
  • Methods for introduction into the oral cavity include direct mixing of RNA with food of the organism.
  • Physical methods of introducing nucleic acids include injection of an RNA solution directly into the cell or extracellular injection into the organism.
  • the agent may be introduced in an amount that allows delivery of at least one copy per cell.
  • a hydrodynamic nucleic acid administration protocol is employed. Where the agent is a ribonucleic acid, the hydrodynamic ribonucleic acid administration protocol described in detail below is of particular interest.
  • the hydrodynamic deoxyribonucleic acid administration protocols described in the art are of interest (Chang et al., J Virol, 75:3469-3473, 2001; Liu et al., Gene Ther, 6:1258-1266, 1999; Wolff et al., Science, 247: 1465-1468, 1990; Zhang et al., Hum Gene Ther, 10:1735- 1737, 1999; and Zhang et al., Gene Ther, 7:1344-1349, 1999).
  • Additional nucleic acid delivery protocols of interest include, but are not limited to: those described in U.S. Patent Nos.
  • the active agent(s) may be administered to the host using any convenient means capable of resulting in the desired reduction of target viral genome amount or load in the infected target cell.
  • the agent can be incorporated into a variety of formulations for therapeutic administration. More particularly, the agents of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.
  • administration of the agents can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, or intracheal administration.
  • the agents may be administered alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
  • the following methods and excipients are merely exemplary and are in no way limiting.
  • the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, corn starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
  • lubricants such as talc or magnesium stearate
  • the agents can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • an aqueous or nonaqueous solvent such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol
  • solubilizers isotonic agents
  • suspending agents emulsifying agents
  • stabilizers and preservatives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • the agents can be utilized in aerosol formulation to be administered via inhalation.
  • the compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
  • the agents can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
  • the compounds of the present invention can be administered rectally via a suppository.
  • the suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
  • Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors.
  • unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
  • the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents, are readily available to the public.
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
  • compositions may be advantageously combined and/or used in combination and/or alternation with other antiviral agents, which are either therapeutic or prophylactic agents, and different from the subject compounds.
  • compositions may also be advantageously combined and/or used in combination with agents that treat conditions often associated with the viral infections, such as anti- HCV agents.
  • administration in conjunction with the subject compositions enhances the efficacy of such agents.
  • the present compounds when combined or administered in combination with other antiviral agents, can be used in certain embodiments in dosages that are less than the expected amounts when used alone, or less than the calculated amounts for combination therapy.
  • Exemplary treatment options for hepatitis C include interferons (e.g., interferon alfa-2b, interferon alfa-2a, and interferon alfacon-1. Less frequent interferon dosing can be achieved using pegylated interferon (interferon attached to a polyethylene glycol moiety thereby significantly improving its pharmacokinetic profile). Combination therapy with interferon alfa-2b (pegylated and unpegylated) and ribavarin has also been shown to be efficacious for some patient populations.
  • interferon alfa-2b pegylated and unpegylated
  • ribavarin has also been shown to be efficacious for some patient populations.
  • RNA replication inhibitors e.g., ViroPharma's VP50406 series
  • antisense agents e.g., ViroPharma's VP50406 series
  • therapeutic vaccines e.g., ViroPharma's VP50406 series
  • protease inhibitors e.g., IL-12
  • helicase inhibitors e.g., IL-12
  • antibody therapy monoclonal and polyclonal
  • the compounds and compositions of the present invention may also be used with agents that enhance the body's immune system, including low-dose cyclophosphamide, thymostimulin, vitamins and nutritional supplements (e.g., antioxidants, including vitamins A, C, E, beta-carotene, zinc, selenium, glutathione, coenzyme Q-IO and echinacea), and vaccines.
  • antioxidants including vitamins A, C, E, beta-carotene, zinc, selenium, glutathi
  • the subject methods find use in the treatment of a variety of different conditions in which the reduction of a target viral genome amount in a target cell or host comprising the same is desired, hi many embodiments, the subject methods find use in the treatment of a host suffering from a viral mediated disease condition.
  • treatment refers to a therapy designed to bring about a reduction or amelioration of the symptoms associated with the condition afflicting the host.
  • a variety of hosts are treatable according to the subject methods.
  • hosts are mammals, animals within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some preferred embodiments, the hosts will be humans.
  • carnivore e.g., dogs and cats
  • rodentia e.g., mice, guinea pigs, and rats
  • primates e.g., humans, chimpanzees, and monkeys.
  • the hosts will be humans.
  • the methods of the present invention may be employed for any viral genome whose abundance in a target cell negatively correlates with a cellular miRNA in that cell.
  • the viral genome is a genome of a virus having an RNA genome, where the virus may be from the family Flaviviridae (e.g., a hepacivirus, such as a hepatitis C virus),
  • the subject invention is employed in methods of treating a host suffering from an HCV mediated disease condition (e.g., associated with infection with non A, non-B hepatitis, NANBH).
  • an effective amount of a miRNA mimic such as an antisense oligo as exemplified below, is administered to the host such that the amount of HCV genome present in the host cells, particularly liver cells, is reduced.
  • compositions containing the miRNA mimics compounds employed in the subject methods can be formulated for oral or parenteral administration for use in the subject methods.
  • the compounds can be admixed with conventional pharmaceutical carriers and excipients (i.e., vehicles) and used in the form of aqueous solutions, tablets, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such pharmaceutical compositions contain, in certain embodiments, from about 0.1 to about 90% by weight of the active compound, and more generally from about 1 to about 30% by weight of the active compound.
  • compositions may contain common carriers and excipients, such as corn starch or gelatin, lactose, dextrose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcram phosphate, sodium chloride, and alginic acid.
  • Disintegrators commonly used in the formulations of this invention include croscarmellose, microcrystalline cellulose, corn starch, sodium starch glycolate and alginic acid.
  • a liquid composition will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s), for example, ethanol, glycerine, sorbitol, non-aqueous solvent such as polyethylene glycol, oils or water, with a suspending agent, preservative, surfactant, wetting agent, flavoring or coloring agent.
  • a suitable liquid carrier for example, ethanol, glycerine, sorbitol, non-aqueous solvent such as polyethylene glycol, oils or water, with a suspending agent, preservative, surfactant, wetting agent, flavoring or coloring agent.
  • a liquid formulation can be prepared from a reconstitutable powder.
  • a powder containing active compound, suspending agent, sucrose and a sweetener can be reconstituted with water to form a suspension.
  • a syrup can be prepared from a powder containing active ingredient, sucrose and a sweetener.
  • a composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid compositions.
  • suitable pharmaceutical carrier(s) include magnesium stearate, starch, lactose, sucrose, microcrystalline cellulose and binders, for example, polyvinylpyrrolidone.
  • the tablet can also be provided with a color film coating, or color included as part of the carrier(s).
  • active compound can be formulated in a controlled release dosage form as a tablet comprising a hydrophilic or hydrophobic matrix.
  • a composition in the form of a capsule can be prepared using routine encapsulation procedures, for example, by incorporation of active compound and excipients into a hard gelatin capsule.
  • a semi-solid matrix of active compound and high molecular weight polyethylene glycol can be prepared and filled into a hard gelatin capsule; or a solution of active compound in polyethylene glycol or a suspension in edible oil, for example, liquid paraffin or fractionated coconut oil can be prepared and filled into a soft gelatin capsule.
  • Tablet binders that can be included are acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (Povidone), hydroxypropyl methylcellulose, sucrose, starch and ethylcellulose.
  • Lubricants that can be used include magnesium stearate or other metallic stearates, stearic acid, silicone fluid, talc, waxes, oils and colloidal silica. Flavoring agents such as peppermint, oil of wintergreen, cherry flavoring or the like can also be used. Additionally, it may be desirable to add a coloring agent to make the dosage form more attractive in appearance or to help identify the product.
  • the compounds of the invention and their pharmaceutically acceptable salts that are active when given parenterally can be formulated for intramuscular, intrathecal, or intravenous administration.
  • a typical composition for intramuscular or intrathecal administration will be of a suspension or solution of active ingredient in an oil, for example, arachis oil or sesame oil.
  • a typical composition for intravenous or intrathecal administration will be a sterile isotonic aqueous solution containing, for example, active ingredient and dextrose or sodium chloride, or a mixture of dextrose and sodium chloride.
  • lactated Ringer's injection lactated Ringer's injection
  • lactated Ringer's plus dextrose injection Normosol-M and dextrose
  • Isolyte E acylated Ringer's injection
  • a co-solvent for example, polyethylene glycol
  • a chelating agent for example, ethylenediamine tetraacetic acid
  • an anti-oxidant for example, sodium metabisulphite
  • the solution can be freeze dried and then reconstituted with a suitable solvent just prior to administration.
  • the compounds of the invention and their pharmaceutically acceptable salts that are active on rectal administration can be formulated as suppositories.
  • a typical suppository formulation will generally consist of active ingredient with a binding and/or lubricating agent such as a gelatin or cocoa butter or other low melting vegetable or synthetic wax or fat.
  • transdermal compositions or transdermal delivery devices
  • Such compositions include, for example, a backing, active compound reservoir, a control membrane, liner and contact adhesive.
  • Transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts.
  • the construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art (See, e.g., U.S. Patent No. 5,023,252).
  • patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.
  • the pharmaceutical composition may contain other pharmaceutically acceptable components, such a buffers, surfactants, antioxidants, viscosity modifying agents, preservatives and the like.
  • these components are well known in the art (See, e.g., U.S. Patent No. 5,985,310).
  • Other components suitable for use in the formulations of the present invention can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed., 1985.
  • the aqueous cyclodextrin solution further comprises dextrose (e.g., about 5% dextrose).
  • kits for practicing one or more of the above-described methods.
  • the kits at least include a miRNA mimic as described above.
  • the kits may also include a pharmaceutically acceptable delivery vehicle, which may be combined with or separate from the miRNA mimic in the kit (e.g., where the two components may be in the same or separate containers in the kit).
  • the subject kits will further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.
  • a suitable medium or substrate e.g., paper on which the information is printed, in the packaging of the kit, in a package insert, etc.
  • a computer readable medium e.g., diskette, CD, etc.
  • a website address accessible via the internet.
  • systems for practicing the subject methods may include one or more pharmaceutical formulations, which include the miRNA mimic.
  • system refers to a collection of components, e.g., active agent, delivery vehicle, etc, present in a single composition or as disparate compositions that are brought together for the purpose of practicing the subject methods.
  • active agent and delivery vehicle brought together and coadministered to a subject, according to the present invention are a system according to the present invention.
  • N normal
  • M molar
  • raM millimolar
  • ⁇ M micromolar
  • mol molecular weight
  • mmol millimoles
  • ⁇ mol micromol
  • nmol nanomoles
  • pmol picomoles
  • g grams
  • mg milligrams
  • ⁇ g micrograms
  • ng nanograms
  • 1 or L L
  • ml milliliters
  • ⁇ l microliters
  • cm centimeters
  • ram millimeters
  • ⁇ m micrometers
  • run nanometers
  • C degrees Centigrade
  • IFN interferon
  • HCV hepatitis C virus
  • miR or miRNA miRNA
  • MOI multiplicity of infection
  • PCR polymerase chain reaction
  • the human hepatoma-derived cell lines Huh7 and their JFH-I replicon-containing subclone were maintained in DMEM with 10% FCS/10mM Hepes, penicillin/streptomycin and 2 mM L-glutamine.
  • the JFH-I full length genomic replicon construct pFGR- JFH-I was obtained from Dr. Takaji Wakita 20 .
  • a Huh7 cell clone that stably replicates the full length genomic HCV RNA was selected and used in these experiment as described previously 21 .
  • the Huh7.5.1c2 cell line was derived from curing replicon-containing Huh-7.5.1 cells by interferon treatment.
  • Primary murine hepatocytes (BaIb C) were collected after collagenase perfusion of the livers for 30 min at 37 0 C.
  • miR array analysis Microarray analysis of total RNA of interferon- stimulated lymphocytes was performed at the Ohio State University Comprehensive Cancer Center Microarray Shared Resource as described 22 .
  • RNA extraction Total RNA was isolated using TRIZOL RNA extraction reagent of Invitrogen, according to the manufacturer's instructions.
  • Real-time PCR Quantitation of HCV genomic RNA was performed using HCV and ⁇ -actin or GAPDH-specific primers as described 15 . Real-time PCR-based quantitation of miRs was performed using miR analysis kits specific for each individual miR (Applied Biosystems) according to the manufacturer's instructions.
  • Recombinant PCR was used to replace the J6CF NS5B-3'UTR region in pCV-J6CF (Yanagi et al., Virology, 262:250, 1999) with the corresponding sequences from JFH-I present in pUC-vJFH (Wakita et al., Nat Med, 11:791, 2005).
  • the NS5B-3 'UTR region of JFH- 1 and an NS5A-NS5B fragment containing a unique Xhol restriction site from J6CF were amplified using primers;
  • XbaJFH GATTACGCCAAGCTTGCATGCCTGCAG, set forth as SEQ ID NO: 12
  • NSSBup CTCCATGTCATACTCCTGGACCGGGGCTC set forth as SEQ ID NO: 13
  • NS5Blo GAGCCCCGGTCCAGGAGTATGACATGGAG set forth as SEQ ID NO: 14
  • XhoJ ⁇ CF AGGTTCC ATCTCTTCC ATGCCCC CCCTCG set forth as SEQ ID NO: 15
  • the two PCR products were mutually extended and amplified using the primers XhoJ ⁇ CF and XbaJFH, and the resultant PCR product was cloned into pGEM-Teasy (Invitrogen, Carlsbad, CA) yielding pTe-J6/JFH and the insert was verified by DNA sequencing.
  • Infectious JFH-I and J6CF/JFH1 viruses were produced by transfection of in vitro synthesized genomic HCV RNA into Huh-7.5.1 cells and virus stocks containing 104-105 focus forming units/ml (ffu/ml) were prepared as described (Zhong et al., Proc Natl Acad Sci U S A, 102:9294, 2005).
  • miRs are a class of small non-coding RNA molecules that function through post-transcriptional regulation of gene expression through a process termed RNA interference (RNAi).
  • RNAi RNA interference
  • These primary microRNAs (pri-miRs) are processed by the enzymes Drosha/DGCR8 into hair-pin loop containing pre-miRs, which are then subject to nuclear export via Exportin 5. Further enzymatic processing of the pre-miRs by Dicer leads to a mature miR duplex that is loaded into the RNA-induced silencing complex (RISC) where the miR guides RISC to complementary mRNAs.
  • RISC RNA-induced silencing complex
  • RISC can inhibit mRNA function by either promoting its cleavage or by inhibiting its translation 3 ' 4 .
  • sequence complementarity in the 6- to 8-base-pair "seed region" at the 5' end of the miR-mRNA heteroduplex appears to determine the specificity of miR-targetRNA interactions 5 .
  • RNAi-based antiviral response in mammalian cells.
  • the potent interferon system has displaced RNAi as the predominant defense against virus infection in mammalian cells '.
  • IFN ⁇ / ⁇ type I interferon-regulated gene products
  • protein kinase R protein kinase R
  • 2 '-5' OA synthase/RNAse L system the adenosine deaminase ADARl or the Mx GTPases are important contributors to the antiviral properties of these cytokines 8 ' 9 .
  • IFN ⁇ / ⁇ might induce cellular miRs that target viral transcripts and thereby utilize RNAi as part of their arsenal against invading viruses has heretofore been unexplored.
  • microarray technology was used to analyze RNA derived from several cell types stimulated with IFN ⁇ / ⁇ or IFN ⁇ for various time periods. The initial screening effort identified ⁇ 30 miRs whose expression levels were increased or attenuated in response to IFN ⁇ / ⁇ or IFN ⁇ . Sequence complementarity analysis of these miRs against viral transcripts or viral genomic RNAs was performed with an initial focus on the crucial seed sequence.
  • HCV is the sole member of the hepacivirus genus of the Flaviviridiae family, and is represented by six major genotypes.
  • the virion harbors a 9.6 kb single- stranded RNA genome of positive polarity with highly invariant 5' and 3' untranslated regions l2 ' 13 .
  • the viral genome is uncoated and serves as a template for the translation of a single large polyprotein, which is subsequently processed by host and viral proteases.
  • the non-structural viral proteins then initiate the synthesis of a negative-strand RNA, which serves as a replication template for the generation of new positive-strand viral genomes ' .
  • IFN ⁇ treatment resulted in a similar level of induction of the prospective antiviral miRs in both cell types as was observed with ISG54, a well characterized IFN ⁇ / ⁇ -regulated gene 14 .
  • Two miRs miR-125 and miR-142 that were found IFN ⁇ -unresponsive in the microarray analysis were included as negative controls.
  • Kinetic and dose-response analysis of the induction of miRs by IFN ⁇ was also performed. Time-course analysis revealed that induction of miR-1 and miR-196, which reached peak concentrations within 30 min, occurs very rapidly and thus even precedes the upregulation of ISG54 (Fig. Ic).
  • miR-122 is specifically expressed in the liver, and previous studies using anti-miRs elegantly demonstrated that miR-122 is essential for HCV replication . Whether miR-122 was also subject to regulation by IFN ⁇ was tested. As shown in Fig. Ie, IFN ⁇ -stimulation of Huh7 cells resulted in a transient, but pronounced (-80%) down-modulation of miR-122 levels. Similar to miR induction, 100 U/ml IFN ⁇ induced maximal attenuation of miR-122 expression, and no additional effect was observed with increased IFN ⁇ concentrations (Fig. Ie).
  • miR-196 was effective against JFHl , but not against J6/JFH containing the "mutant" target site.
  • miR-448 only inhibited the replication of JFHl containing the 'correct" target site, but was ineffective against J6/JFH (Fig. 3b).
  • Introduction of a compensatory single nucleotide change into miR-196 and miR-448 (designated miR-196* and miR-448*) matching their seed sequence to J6/JFH yielded a reversed efficacy profile compared to the wild-'type miRs when they were tested against the chimeric viruses.
  • IFN ⁇ treatment leads to a >90% reduction in the amount of viral HCV replicon RNA, which is unaffected by transfected non-specific control anti-miRs.
  • introduction of the anti-miR mix or of the miR-122 mimic separately attenuated the IFN ⁇ effect to -75% inhibition.
  • Co-transfection of the anti-miR mix and the miR-122 mimic further reduced the efficacy of IFN ⁇ to -50%, indicating that modulation of the expression levels of the identified miRs plays an important, albeit not exclusive role in the antiviral effects of IFN ⁇ against HCV.
  • Results shown as p-values for a paired T-test.
  • IFN ⁇ up- regulates several cellular miRs, which are capable of inhibiting HCV replication and infection.
  • down-regulation of miR-122 in response to IFN ⁇ further contributes to the antiviral effects of this cytokine.
  • This example provides methods for evaluating the microRNA compositions of the present invention in rodent and nonhuman primate models of Hepatitis C virus infection.
  • the methods of the present example are based upon published animal studies of RNAi (See, e.g., WO 2007/076328, and Elmen et al., Nature, 452:896-899,
  • a method for expressing hepatitis C virus in an in vivo animal model has been developed (Vierling, International PCT Publication No. WO 99/16307). Briefly viable, HCV infected human hepatocytes are transplanted into a liver parenchyma of a scid/scid mouse host. The scid/scid mouse host is then maintained in a viable state, whereby viable, morphologically intact human hepatocytes persist in the donor tissue and hepatitis C virus is replicated in the persisting human hepatocytes.
  • This model provides an effective means for the study of HCV inhibition by microRNA administration in vivo.
  • GBV-B is very closely related to human hepatitis C virus and causes hepatitis in tamarins and marmosets.
  • GBV-B infection of tamarins and marmosets are suitable nonhuman primate models for testing antiviral compounds and vaccines for HCV infection.
  • the GBV-B model is an appropriate system to test whether the microRNA-based therapies of the present invention are likely to work on humans chronically infected with HCV.
  • two animals are inoculated with GBV-B and treatment with the raicroRNA compositions of the present invention is initiated one day post infection. Another two animals are inoculated with GBV-B and are untreated to serve as negative controls.
  • the animals are monitored to determine the effect of the therapy of GBV-B infection. Blood draws are performed over the course of the study to determine viral titers. Dosing of microRNA compositions in the treated animals is repeated at days 1, 3, and 7 after inoculation at day 0. The treated animals are contemplated to have a measurable inhibition of GBV-B over a 1 -4 week time course as compared to the untreated control animals. In addition, several animals with established GBV infections as treated with the microRNA compositions of the present invention at days 28, 31, and 35 post-infection. These animals are contemplated to show a measurable following dosing, as compared to historic untreated controls.
  • the microRNA compositions of the present invention are evaluated in HCV-infected chimpanzees.
  • the compositions are administered by IV.
  • the chimpanzees to be used are selected from a group of HCV chronic animals.
  • the study is conducted in two phases: pharmacokinetics and efficacy.
  • the pharmacokinetics portion of the study is conducted in two non-HCV-infected animals. Blood samples are obtained at times: Omin, 15min, 30min, 24hr, and days 3, 7 and 14. Liver biopsies are obtained at 24 hr and 14 days.
  • the efficacy involves testing of the antiviral compound in 2 or more HCV infected chimpanzees.
  • Animals receive 4 weekly IV injections with the antiviral microRNA formulations. Blood samples are obtained at -4, -2, and 0 weeks, then weekly for 6 weeks, and then every other week for 4 additional weeks. Liver biopsies are obtained at -4 weeks and +4 weeks (one week after last injection). The animals are monitored for blood chemistries and complete blood counts (CBC) at all bleeds. At the sign of any serious adverse effects, treatment is stopped. For each animal, multiple blood samples are taken to monitor the viral RNA levels in the serum. Liver needle biopsies are requested at two time points for analysis of viral RNA load in the liver, level of microRNA targeted to liver, and changes in liver gene expression. Viral RNA is monitored by real time quantitative RT-PCR.

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Abstract

La présente invention concerne la réduction de l'accumulation de génomes viraux dans une cellule cible. La présente invention concerne en particulier des compositions et des méthodes pour combattre une infection virale par ARN interférence. Plus spécifiquement, la présente invention concerne des mimétiques de micro-ARN cellulaires destinés au traitement de sujets infectés par un virus.
PCT/US2008/074513 2007-08-27 2008-08-27 Micro-arn utilisés pour inhiber la réplication virale WO2009029681A2 (fr)

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WO2010042683A1 (fr) * 2008-10-08 2010-04-15 The Charlotte-Mecklenburg Hospital Authority D/B/A Carolinas Medical Center Traitement de l'infection par le virus de l'hépatite c avec une surexpression de micro-arn-196
EP2191834A1 (fr) * 2008-11-26 2010-06-02 Centre National De La Recherche Scientifique (Cnrs) Compositions et procédé pour traiter des infections à rétrovirus
WO2010060967A2 (fr) * 2008-11-26 2010-06-03 Centre National De La Recherche Scientifique (C.N.R.S) Compositions et procédés pour traiter des infections rétrovirales
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US8771226B2 (en) 2010-02-18 2014-07-08 Bio2Medical, Inc. Vena cava filter catheter and method
TWI454577B (zh) * 2012-12-19 2014-10-01 Univ Kaohsiung Medical 預測干擾素療效之方法及套組
EP2757157A1 (fr) * 2013-01-17 2014-07-23 Ecole Polytechnique Federale de Lausanne (EPFL) Modulation de mitophagie et son utilisation
WO2014111876A3 (fr) * 2013-01-17 2014-10-30 Ecole Polytechnique Federale De Lausanne (Epfl) Modulation de la mitophagie et son utilisation

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